This application seeks to address the impact of bacterial colonization and persistence in chronic wounds. The formation of biofilms has clearly been linked to chronic and persistent bacterial infections. This considerably delays and complicates wound healing. Unlike acute bacterial infections, which are often cleared by the host, biofilm-related chronic infections are not easily resolved even with high dose antibiotics and intact immunity. The bacterial pathogens Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus, which are the focus of this application, cause an array of biofilm-related clinical diseases including persistent airway infections, burn wound infections, endocarditis, and surgical site infections. Unresolved infected wounds also contribute to nosocomial persistence and the spread of bacteria in health care settings. This proposal will use state-of-the art molecular and genomic approaches to define genes and pathways essential for P. aeruginosa persistence in chronic wounds in monoculture as well as in the presence of S. aureus and/or A. baumannii. Investigating this hypothesis will allow us to learn new P. aeruginosa biology and aid future management and treatment of chronic infections. Aim 1 will identify genes and pathways necessary for P. aeruginosa during a chronic wound infection. Aim 2 will identify genes and pathways necessary for P. aeruginosa during polymicrobial chronic wound infections. The development of a chronic infection model that can be sampled over time provides a relevant, unique, and novel approach for examining the effects of biofilm formation in the host. Ultimately information gathered will aid the treatment of an array of chronic infections including highly prevalent persistent wound infections.